Daphnane Diterpenoids from Trigonostemon lii and Inhibition Activities Against HIV-1

Abstract Natural products are the important source for the discovery of more potent anti-HIV agents. In this study, six daphnane diterpenoids including three unreported structures were isolated from Trigonostemon lii, which showed significant activities against HIV-1 strains replication in the nanomolar/picomolar range. Meanwhile, these diterpenoids significantly inhibited the fusion of H9/HIV-1 IIIB cells with uninfected C8166 cells, with the EC50s from 1.06 to 8.73 ng/mL, and did not show any inhibition activities against HIV-1 reverse transcriptase. Moreover, all of the diterpenoids shows significant inhibitions against T20-resistan HIV-1 strains, PNL4-3gp41(36G)V38E, N42S and pNL4-3gp41(36G)V38A, N42T. The results revealed that the six diterpenoids could be a new type of potential lead candidate as an HIV entry inhibitor, particularly for those infected by T20-resistant variants. Graphic Abstract Electronic supplementary material The online version of this article (10.1007/s13659-020-00231-7) contains supplementary material, which is available to authorized users.


Introduction
According to the latest report on the global AIDS epidemic, infections with the human immunodeficiency virus 1 (HIV-1) remain a global threat to public health [1]. Drug treatment for controlling viral loads and for prolonging patients' lives is the main therapy for HIV-1 infections. Highly active anti-retroviral therapy now has shown significant synergistic effects on prolonging the lifetime and decreasing the mortality of patients, the issues such as toxicity, viral reservoirs and drug resistance have led to a subsequent crisis in the management of HIV/AIDS patients [2]. The potent drugs may fail at a later stage. In the light of these considerations, new classes of drugs against HIV are in urgent need to develop.

3
The process of HIV-1 entry into host cells considerable potential for therapeutic intervention, with viral entry proceeding through multiple sequential steps involving attachment, coreceptor blinding, and fusion [3]. The discovery and characterization of new anti-HIV agents of those steps to be the host remain priority.
Natural products have served as the important leads for the discovery of more potent anti-HIV agents [4,5]. However, very few natural products were discovered as having anti-HIV-1 potential in the nanomolar/picomolar range [4,6]. Here, we reported six daphnane diterpenoids (compounds 1-6) including three unreported ones [trigonolactones B (1), D (2) and E (3)] with significant anti-HIV-1 activity from Trigonostemon lii [7] (Fig. 1). These compounds not only exhibited strong inhibition on HIV replication with EC 50 values of 0.59-8.22 ng/mL and SI values of 1811-29,610, but also displayed significantly inhibited the fusion of H9/HIV-1IIIB cells with uninfected C8166 cells with EC 50 values of 1.06-8.73 ng/mL, while did not show any inhibition activity against HIV-1 reverse transcriptase. More important, these compounds still displayed significant inhibitions against T20-resistant HIV-1 strains, pNL4-3gp41(36G)V38E, N42S and pNL4-3gp41(36G)V38A, N42T. The structures of these compounds were established by spectroscopic approach including 1D, 2D NMR and HRMS technology.  Table 1). The above data suggested that compound 1 and the previously identified molecule trigocherriolide B [8,9] share a similar scaffold, except that one methylene signal at δ H 5.17 (s), 5.07 (s) replaced the C-19 methine signal at δ H 6.07 (s) in latter. Considering the molecular weight of 1 is 34 units less than that of trigocherriolide B, the compound should be 19-dechloro form of trigocherriolide B. The further HMBC cross peaks from H-3 to C-1 and C-19, H 2 -19 to C-2 and C-1, and H-1 to C-19 and C-2 located the exo-methylene at C-19. Thus, the gross structure of trigonolactone B (1) was established as shown (Fig. 2).

Structure Elucidation
The relative configuration of 1 was elucidated by the ROESY experiment and compared with known compounds. The ROESY spectrum exhibited strong correlations of the axial H-11 to H-8 and H-12, orienting H-8 and H-12 on the β-orientation. The β-configurations of H-14, OH-4 and H-7 were established on the correlations of H-8 to H-7, H-14 and OH-4, meanwhile revealed that the 9, 12, 14-orthobenzoate was α-directed. The mutual ROESY correlations between H-3 and H-5 oriented the same α-configuration. Thus, the structure of 1 was elucidated as shown (Fig. 3).
The molecular formula of trigonolactone D (2) was indicated to be C 45      T20 is the only FDA-approved first-generation HIV fusion inhibitor, which is being used for treatment of HIV/AIDS patients who have failed to respond to current antiretroviral drugs. Unfortunately, many patients are now failing to respond to enfuvirtide because it rapidly induces drug resistance in vitro and in vivo [11][12][13]. Thus, the inhibition assay of microtiter syncytium formation of the two T20-resistant HIV-1 strains, pNL4-3gp41(36G)V38E,N42S and pNL4-3gp41(36G)V38A,N42T in C8166 cells, were used to evaluate anti-HIV activity, respectively. All of the compounds showed significant inhibitoies with EC 50 s of 3.30, 2.72, 4.43, 2.97, 2.88 and 3.74 ng/mL for the former (Fig. 5a), and EC 50 s of 2.60, 5.83, 3.19,1.85, 3.43 and 3.81 ng/mL for the later (Fig. 5b) (EC 50 of T20 > 1000 ng/mL).

Mechanisms of Action
To address the action mechanisms, further experiments were carried out. HIV reverse transcriptase (RT) plays a very important role in the HIV replication, so the anti-HIV-1 RT activities of 1-6 were evaluated. The results demonstrated  (Fig. 4b), and when concentrations were 40 ng/mL, they inhibited about 90% of syncytia formation.
To further address action mechanisms of 1-6, more experiments were carried out. In co-cultivation assay, those compounds effectively inhibited the fusion of H9/HIV-1 IIIB cells with uninfected C8166 cells but in RT study, it was found that they can't inhibit HIV-1 RT activity at concentration as high as 300 μg/mL. These data supported that those compounds interfered HIV entry target cells possible.
Trigonothyrins A, B, C, D, F and G were tested for inhibitory activity against HIV-1 using the same method mentioned above [14,15]. Only trigonothyrins C and F showed modest anti-HIV-1 activitise with EC 50 s of 2.19 and 0.13 μg/ mL, and TI value of > 91.3 and 75.1 [14]. Analysis of those structures implied the macrolactone moiety and conjugative A ring appeared to be contributed for much higher antiviral activity. Compounds 19-Chlorine substituted 4, 5 and 6 was less active than 1, 2 and 3 respectively, indicating that the substitution pattern is not good for the activity. When 2-hydroxyl group were introduced at C-17, the bioactivity increased significantly, indicating the substitution at C-17 contribute to the improvement of inhibitory activity. We found compounds 1-6 possesses low cytotoxicities to C8166 and exhibited potent anti-HIV activities. It not only inhibited replication of HIV-1 laboratory strains (HIV-1 IIIB ), but also inhibited T20-resistant strains (pNL4-3 gp41(36G)V38E,N42S and pNL4-3 gp41(36G)V38A,N42T ) that those compounds showed strong inhibitory activities against all T20-resistant strains with EC 50 values about 10 ng/mL. It was interesting that compounds 1-6 were effective in these T20-resistant strains and the EC 50 s were similar in HIV-1IIIB. This result implied that FuSC-1 inhibit HIV with the mechanism different to HIV fusion inhibitor T20 and suggest that this class of compound can be further developed as an alternative entry inhibitor for treatment of patients with HIV-1/AIDS, those infected with T20-resistant variants.

General Experimental Procedures
Optical rotation was carried out on a Perkin-Elmer model 241 polarimeter. IR spectra were measured in a Bio-Rad FTS-135 spectrometer with KBr pellets, whereas UV data were measured using a UV-210A spectrometer. Electrospary ionization-mass spectrometry (ESI-MS) and high-resolution (HR) ESI-MS were recorded with an APIQSTAR Pulsar 1 spectrometer (Advanced Biomics, Los Angeles). The 1D and 2D NMR spectra, including COSY, ROESY, HMBC, and HSQC experiments, were acquired at room temperature using a Bruker AM-400 and DRX-500 spectrometers operating at 400 and 500 MHz ( 1 H) and 100 and 125 MHz ( 13 C), respectively, with tetramethylsilane (TMS) as an internal standard. Multiplicities were determined using the DEPT pulse sequence. Column chromatography was performed on Si heat-inactivated newborn calf serum (Gibco). The HIV-1 IIIB virus was obtained from MRC, AIDS Reagent Project, UK. The 50% HIV-1 tissue culture infectious dose (TCID50) was determined and calculated by the Reed and Muench method. Virus stocks were stored in aliquots at − 70 °C [16].

Extraction and Isolation
The  (18)

MTT-Based Cytotoxicity Assay
Cellular toxicity of compounds was assessed by MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations of compounds in triplicate and incubated at 37 °C in a humid atmosphere of 5% CO 2 for 3 days. Twenty microliters of MTT reagent (5 mg/mL in PBS) was added to each well, then incubated at 37 °C for 4 h, 100 μL of 50% DMF-20% SDS was added. After the formazan was dissolved completely, the plates were read on a Bio-Tek ELx 800 ELISA reader at 595 nm/630 nm (A595/630). The cytotoxic concentration that caused the reduction of viable cells by 50% (CC 50 ) was calculated from dose-response curve [17].

Syncytia Assay
In the presence of 100 μL various concentrations of compounds, C8166 cells (4 × 10 5 /mL) were infected with virus (HIV-1 IIIB ) at a multiplicity of infection (M.O.I) of 0.06. The final volume per well was 200 μL. Control assays were performed without the testing compounds in HIV-1 IIIB infected and uninfected cultures. AZT was included as positive control. After 3 days of culture, the cytopathic effect (CPE) was measured by counting the number of syncytia (multinucleated giant cell). Percentage inhibition of syncytia formation was calculated and 50% effective concentration (EC 50 ) was calculated [18,19].

Co-cultivation Assay
C8166 cells (3 × 10 4 ) co-cultured with 1 × 10 4 virus (HIV-1 IIIB ) infected H9 cells in the presence or absence of the compound with various concentrations at 37 °C in a humidified atmosphere of 5% CO 2 . T20 was used as positive control. After 6 h incubation, the number of syncytia was scored under an inverted microscope [18].

RT (Reverse Transcriptase) Assay
HIV-1 RT activity was measured by ELISA RT kit (Roche) using a commercially available kit according to the protocol provided by the manufacturer. Samples were incubated with DIG-labeled-reaction mixture at 37 °C for 15 h. Anti-DIG-POD solution was added afterward followed by substrate ABTS. The absorbance at 405/490 nm (A405/490) was determined in the ELISA reader [20].